Synthesis of new 9-hydroxy-α- and 7-hydroxy-β-pyran naphthoquinones and cytotoxicity against cancer cell lines

Article abstract of DOI:10.1039/c1ob05209h

A synthetic method to obtain α- and β-pyran naphthoquinones 10 and 11 with a hydroxyl substituent on the aromatic ring was developed. Two series of α- and β-pyran naphthoquinones were obtained from the 8-hydroxy-lawsone, and their anticancer properties we

Full text of DOI:10.1039/c1ob05209h

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PAPER
Synthesis of new 9-hydroxy-a- and 7-hydroxy-b-pyran naphthoquinones and
cytotoxicity against cancer cell lines†
David R. da Rocha,*a Ana C. G. de Souza,a Jackson A. L. C. Resende,b Wilson C. Santos,c
Evelyne A. dos Santos,d Cla´udia Pessoa,d Manoel O. de Moraes,d Let´ıcia V. Costa-Lotufo,d
Raquel C. Montenegrod and Vitor F. Ferreira*a
Received 7th February 2011, Accepted 7th March 2011
DOI: 10.1039/c1ob05209h
A synthetic method to obtain a- and b-pyran naphthoquinones 10 and 11 with a hydroxyl substituent
on the aromatic ring was developed. Two series of a- and b-pyran naphthoquinones were obtained
from the 8-hydroxy-lawsone, and their anticancer properties were evaluated against four tumor cell
lines. In general, the new compounds displayed good activity, possibly indicating that these compounds
have increased pro-oxidant capacity. The 9-hydroxy-a-lapachone and 7-hydroxy-b-lapachone analogues
of the natural products a-lapachone and b-lapachone were successfully produced by this methodology.
Indeed, several quinones, such as doxorubicin, mitomycin, and
mitoxantrone, have become medicines that are still used clinically
The International Agency for Research on Cancer (IARC/WHO)
in the therapy of solid cancers.
Lapachol (1) is a natural naphthoquinone that occurs in the
worldwide by the year 2010, and the incidence of cancer is
grain of several wooden trees of the Bignoniaceae family. Lapachol
Introduction
predicted that cancer would be the leading cause of death
still increasing, particularly in developing countries.1 Although
considerable insight has been gained into the mechanisms by
which some chemicals affect cellular growth, and this knowledge
has been used for the design of new chemotherapeutic drugs, new
and more selective lead compounds are still needed. In this regard,
quinones exhibit important cytotoxic activity against cancer cells.2
reduces cancer metastasis3 and acts upon DNA topoisomerases,
enzymes that are essential for the integrity of the DNA molecule.4
Two other important pyranaphthoquinones, the a- (2) and b-
lapachones (3), are isolated as minor components of heartwood
from Lapacho trees (Fig. 1). The characteristics of these com-
pounds have been extensively discussed in the literature.5
In 1984, Boorstein and Pardee reported that b-lapachone
increases the lethality of human fibroblasts.6 Pursuing this line
of research, Pardee and coworkers found that b-lapachone sup-
pressed HIV-1 replication in both acute and chronic infection7
and also inhibited the activity of topoisomerase by blocking
the formation of the topoisomerase I-DNA cleavable complex.8
Following these seminal works, hundreds of reports on the activity
of b-lapachone against various tumor cells were published in
just over fifteen years. For example, anticancer activities were
reported against human cancer cell lines from leukemia9 and
prostate,10 malignant glioma,11 hepatoma,12 colon,13 breast,14
lung15 and pancreatic cancers.16 b-Lapachone (3) has been reported
aUniversidade Federal Fluminense, Departamento de Qu´ımica Orgaˆnica,
Outeiro de Sa˜o Joa˜o Batista, s/n◦, Centro, Nitero´i, 24020-141, Rio de
Janeiro, Brazil
bUniversidade Federal Fluminense, Departamento de Qu´ımica Inorgaˆnica,
Outeiro de Sa˜o Joa˜o Batista, s/n◦, Centro, Nitero´i, 24020-141, Rio de
Janeiro, Brazil
cUniversidade Federal Fluminense, Faculdade de Farma´cia, Rua Ma´rio
Viana, 523, Santa Rosa, Nitero´i, 24241-000, Rio de Janeiro, Brazil
dUniversidade Federal do Ceara´, Laborato´rio de Oncologia Experimental,
Departamento de Fisiologia e Farmacologia, CEP, 60431-970, Fortaleza,
CE
† CCDC reference numbers 811449 and 811450. For crystallographic data
in CIF or other electronic format see DOI: 10.1039/c1ob05209h
Fig. 1 Important natural naphthoquinones.
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to increase the lethality of human cancer cells in association
with other drugs such as taxol,17 mitomycin C (ovarian)6 and
paclitaxel.18 Boothman and coworkers discovered another re-
markable property of b-lapachone (3), an ability to act as a
co-adjuvant in killing human cancer cells during radiotherapy
treatment. It appears to inhibit sub-lethal radiation damage
repair.19 Since then, this activity has been exploited by several
Results and discussion
Chemistry
The first step in the preparation of the desired a- and b-pyran
naphthoquinones or a- and b-lapachone derivatives 10a–g and
11a–g, was the oxidation of 1,5-naphthalene diol (5) with HIO4
to produce juglone (6) in 85% yield. The next step involved a
Michael addition of p-thiocresol to 6 that produced two isomeric
adducts, 7 and 8 (Scheme 1 and Fig. 2). Although a previous
report of this reaction28 only describes the synthesis of product 8,
we obtained a mixture of isomers. Purification of the mixture by
column chromatography on silica gel using gradient elution with
hexane and toluene furnished adducts 7 and 8 in 40% and 47%
yields, respectively.
researchers and is currently a medical procedure under patent.20
A
new modality of radiotherapy using gold nanoparticles containing
b-lapachone for radiosensitization that improved radiotherapeutic
efficacy was reported very recently.21 A modified cyclodextrin
host–guest complex of b-lapachone associated with gemcitabine,
named ARQ501, is currently undergoing multiple Phase II clinical
trials for use against pancreatic cancer and adenocarcinoma.22
Because b-lapachone displays interesting anticancer activities
and is readily available from natural sources, it has become a
favored structure in medicinal chemistry. Many synthetic routes
for obtaining 3 have been developed, and several derivatives
have been prepared and biologically evaluated. However, a- and
b-lapachones substituted in the aromatic ring have not been
well studied because they are difficult to obtain from other
natural pyranaphthoquinones by aromatic electrophilic substi-
tution reactions in the aromatic ring. These compounds can be
isolated as natural products from several sources. For example, a-
caryopterone (4), isolated from Caryopteris clandonensis (Fig. 1),
exhibits molluscicidal and radical scavenging activity.23 Giles24 and
Oliveira25 developed syntheses of a-caryopterone (4) and, more re-
cently, Padro´n26 reported the synthesis of 5-hydroxy-lapachol (1b).
The ability of naphthoquinones to cause damage to DNA and
peroxidation of lipids is closely related to the pro-oxidant action
of the semiquinone radical. Yoshino and co-workers27 found that
naphthoquinones with a hydroxyl group on the benzene moiety
stimulated lipid peroxidation and the formation of adducts with
DNA.
In this paper, we report the preparation of a- and b-pyran
naphthoquinones 10a–h and 11a–h, with a hydroxyl group at
position 7 or 9 of the aromatic ring, respectively, via ortho-
quinone methide intermediates (o-QMs) generated in situ by
Knoevenagel condensation of 2,8-dihydroxy-1,4-naphthoquinone
(9) with formaldehyde, followed by a hetero Diels–Alder reaction
with substituted styrenes in ethanol under microwave irradiation.
We also report the cytotoxic activities of these a- and b-lapachone
derivatives against four neoplasic cancer cell lines, MDA-MB435
(breast/melanoma), HL-60 (leukemia), HCT-8 (colon) and SF-
295 (central nervous system), and toward one normal cell type,
murine fibroblast L-929. All cell lines originated from the National
Institute of Health, Bethesda, Maryland.
Fig. 2 Ortep drawing of naphthoquinones 7 and 8, respectively.
It was impossible to differentiate the structures of 7 and 8 on
1
the basis of their H and 13C NMR spectra, but we were able to
unambiguously determine the structures using X-ray diffraction.
Hydrolysis of 8 under basic conditions produced naphtho-
quinone 9 in 90% yield. The removal of the p-thiocresol group
1
was confirmed by H and 13C NMR spectra. The final step in
the preparation of the target a-and b-pyran naphthoquinones
10a–h and 11a–h was a Knoevenagel reaction followed by hetero
Diels–Alder cycloaddition reactions under microwave irradiation
Scheme 1 Synthesis of 1,4-naphtoquinones 7 and 8.
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using several styrenes as hetero-dienophiles. This methodology
was first reported by Ferreira and Ventura Pinto,29 in the 1980’s,
improved by Nair and co-workers,30 and more recently, expanded
by Ferreira and co-workers (Scheme 2).31 In all reactions, the
a-pyran naphthoquinone 10a–h was the major product in an
approximate ratio of 2 : 1. These results are consistent with those
described in the literature, which indicate that the a-isomer always
forms in greater proportion. These isomers were separated by
column chromatography using silica gel and hexane–ethyl acetate
eluent mixtures of increasing polarity.
Biological assay
Compounds 10a–h and 11a–h were tested in vitro against four
cancer cell lines and one normal cell line, murine fibroblast L-929,
using the MTT assay with doxorubicin as a positive control.32
Concentrations (in mM) that induced 50% inhibition of cell growth
(IC50) are reported in Table 1.
The introduction of the hydroxyl group in the analogues of
9-hydroxy-a-lapachone (10a,10e, 10g and 10h) increased their
activity against all cancer cell lines relative to a-lapachone (2),
Scheme 2 Synthesis of pyran naphthoquinones 10a–h and 11a–h.
Table 1 Cytotoxic activity expressed as IC50 in mM of compounds against cancer cell lines
Compounds
Entry
Cancer cell linesa
MDA-MB435
Erythrocytes
HL-60
HCT-8
SF295
(ED50 – mg mL-1)
1
2
3
4
5
6
7
8
Doxob
2
0.88 (0.62–1.21)
Inactive
0.25 (0.16–0,33)
6.77 (4.11–9.40)
Inactive
Inactive
Inactive
2.47 (2.07–2.87)
6.80 (4.40–9.21)
2.90 (2.24–3.76)
0.11 (0.11–0.12)
0.15 (0.10–0.20)
0.15 (0.09–0.22)
0.17 (0.15–0.19)
0.21 (0.20–0.27)
0.53 (0.35–0.80)
0.47 (0.46–0.53)
0.31 (0.27–0.36)
0.03 (0.02–0.04)
Inactive
1.65 (1.49–1.78)
9.38 (7.08–11.69)
Inactive
Inactive
Inactive
8.73 (5.89–11.56)
12.05 (6.27–17.82)
6.03 (5.34–6.73)
1.30 (1.11–1.50)
3.84 (2.28–6.52)
3.28 (1.56–6.84)
3.40 (2.52–4.61)
3.21 (2.65–3.85)
2.85 (1.28–6.27)
2.56 (2.15–3.03)
1.94 (1.63–2.28)
0.06 (0.04–0.08)
Inactive
0.83 (0.74–0.87)
12.44 (9.73–15.14)
Inactive
Inactive
Inactive
10.48 (8.73–12.24)
13.84 (11.51–16.17)
6.72 (5.57–7.86)
1.40 (1.14–1.73)
3.30 (1.63–6.67)
3.28 (2.25– 4.71)
2.49 (2.11–2.90)
2.81 (2.31–3.39)
5.88 (2.59–13.29)
3.06 (2.72–3.40)
2.17 (2.01–2.32)
0.41 (0.29–0.44)
Inactive
0.91(0.74–1.11)
Inactive
Inactive
Inactive
Inactive
3.54 (3.02–4.07)
ND
5.63 (4.49–6.78)
2.15 (1.86–2.51)
0.99 (0.78–1.22)
2.34 (1.78–3.06)
2.84 (2.26–3.55)
3.21 (2.25–4.56)
6.69 (3.75–11.98)
4.71 (3.78–5.93)
2.52 (2.13–3.02)
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
3
10a
10b
10c
10d
10e
10g
10h
11a
11b
11c
11d
11e
11f
11g
11h
9
10
11
12
13
14
15
16
17
18
a Data are presented as IC50 values and 95% confidence intervals obtained by nonlinear regression for all cell lines from three independent experiments.
b Doxorubicin (Doxo) was used as positive control. Only compounds with an IC50 value lower than 5 mg mL-1 for at least one cell line were considered
active.
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which is inactive, but they were less active than the 7-hydroxy-b-
lapachones (11a–h). Nevertheless, it seems that these compounds
were more pro-oxidant than the parent structure.
at 130(2)K. Compounds 10a–g and 11a–g were prepared using a
CEM microwave irradiation Discover 1. Compounds 10 h and
11 h were prepared in a Berghof High-Pressure Reactor BR-300.
Compound 6 was prepared according to literature data.34
The introduction of the hydroxyl group at C-7 of the pyran
naphthoquinones promoted changes in the pattern of activity of
these substances compared with b-lapachone (3) and doxorubicin.
In general, the b-pyran naphthoquinones 11a–h were more
active than doxorubicin on melanoma cells (MDA-MB435). The
presence of halogens on 11b, 11d and 11e increased the selectivity
for MDA-MB435 without any significant difference between the
three compounds. With the exception of 7-hydroxy-b-lapachone
(11h, entry 18), which was less active than b-lapachone (3, entry
3) against all cell lines, the b-pyran naphthoquinones 11a–h
(entries 11–18) were more active and selective against melanoma
cancer cells (MDA-MB435) than b-lapachone (3) and doxorubicin.
Compound 11a was particularly effective, displaying 8-fold greater
activity than doxorubicin. None of the compounds exhibited lytic
effects against mouse erythrocytes.
8-hydroxy-2-(p-tolylthio)naphthalene-1,4-dione (8) and
5-hydroxy-2-(p-tolylthio)naphthalene-1,4-dione (7)
A round-bottom flask equipped with a magnetic stirring bar was
loaded with 6 (5.4 mmol) dissolved in ethyl alcohol (40 mL).
Next, 4-methylbenzenethiol (5.4 mmols) was dissolved in ethyl
alcohol (10 mL) and added to the reaction mixture. The reaction
proceeded for 4 h, after which the ethanol was removed under
reduced pressure. The residual crude product was purified via
silica-gel chromatography, using a gradient mixture of hexane and
toluene to yield 7 (40%) and 8 (47%). The physical characteristics
of these compounds were identical to previous reports.28
Crystal structure determinations
Conclusion
Crystal data 7: C17H12O3S, Mr = 296.34, monoclinic, P21/◦n, a =
˚
˚
˚
9.713 (2)A, b = 11.179 (2)A, c = 13.663 (3)A, b = 109.59(3) , V =
A synthetic method to obtain a- and b-pyran naphthoquinones
10a–h and 11a–h with a hydroxyl substituent on the aromatic ring
was developed. Two series of a- and b-pyran naphthoquinones
were obtained from the 8-hydroxy-lawsone, and their anticancer
properties were evaluated against four tumor cell lines. In general,
the series of 9-hydroxy-a-lapachones displayed greater activity
than a-lapachone (2), possibly indicating that these compounds
have increased pro-oxidant capacity. The derivatives of 7-hydroxy-
b-lapachone (11a–h) showed excellent results against melanoma
cancer cells (MDA-MB435). Compounds 11a (IC50 0.11 mM), 11b
(IC50 0.15 mM), 11c (IC50 0.15 mM), 11d (IC50 0.17 mM) and 11f
(IC50 0.23 mM) were particularly effective. These results demon-
strate that the introduction of a hydroxyl group on the aromatic
ring increased the selectivity and activity of these compounds
for MDA-MB435 cells and suggest that other substituents may
also increase these activities if located on other positions of the
aromatic ring. Finally, the 9-hydroxy-a-lapachone (10h) and 7-
hydroxy-b-lapachone (11h) analogues of the natural products a-
lapachone (2) and b-lapachone (3) were successfully produced by
this methodology.
3
-1
˚
1397.7(6)A , Z = 4, T = 293(2) K, m = 0.24 mm , 8729 reflections
measured, 2473 unique (Rint = 0.0920). The final R1(F2) was 0.0509
for all data. CCDC reference number 811450.
Crystal data 8: C17H12O3S, Mr = 296.34, monoclinic, P21/c, a =
◦
˚
˚
˚
˚
8.9234(7)A, b = 8.6499(7)A, c = 17.8425(8)A, b = 103.82(1) , V =
3
-1
1337.3(2)A , Z = 4, T = 130(2) K, m = 2.21 mm , 3868 reflections
measured, 1872 unique (Rint = 0.020). The final R1(F2) was 0.0345
for all data. CCDC reference number 811449.
2,8-dihydroxy-1,4-naphthoquinone (9)
A round-bottom flask equipped with a magnetic stirring bar was
loaded with 8 (1.5 mmol) dissolved in ethyl alcohol (24 mL) and
an aqueous solution of NaOH (2 N, 12 mL). The mixture was
heated under reflux for 3 h. Water (30 mL) was added, and
the mixture was cooled in ice bath and neutralized with 4 N
H2SO4. The ethanol was removed under reduced pressure, and
the mixture was extracted with ethyl acetate. The organic layer
was washed with water, dried over anhydrous sodium sulfate,
filtered and concentrated under reduced pressure. The residual
crude product was purified via silica-gel chromatography, using a
gradient mixture of hexane and ethyl acetate to yield 9 in 90%. The
physical characteristics of 9 were identical to previous reports.35
Experimental
Chemical synthesis
General remarks. Melting points were obtained on a Fisher–
Johns melting point apparatus and are uncorrected. Analytical
grade solvents were used, and the solvents were previously purified
as described in the literature.33 Column chromatography was
performed on silica-gel (Acros Organics 0.035–0.070 mm, pore
diameter ca. 6 nm). Infrared spectra were recorded on an ABB
FTLA2000-100 spectrometer. 1H NMR spectra were collected
in CDCl3 on a Varian Unity Plus 300 instrument. Elemental
analysis was performed with a Perkin-Elmer CHN 2400. X-ray
diffraction of compound 7 was performed on a Bruker-Kappa-
General procedures for preparing 10a–g and 11a–g
A 10 mL microwave tube was loaded with 9 (2.6 mmol),
paraformaldehyde (8 mmols), the appropriate styrene (3 mmols)
and anhydrous ethanol (5 mL) and irradiated for 15 min. The
internal temperature reached 150 ◦C. The solvent was evaporated
under reduced pressure, and the crude mixture was extracted with
ethyl acetate (30 mL). The organic layer was washed with water
(3 ¥ 20 mL), dried over anhydrous sodium sulfate, filtered and then
concentrated under reduced pressure. The residual solid product
was purified by column chromatography on silica-gel and eluted
with an increasing polarity gradient mixture of hexane and ethyl
acetate (9/1 to 7/3).
˚
CCD diffractometer using Mo-Ka radiation (l = 0.71069 A) at
room temperature, and compound 8 was analyzed on a Oxford
˚
Diffraction Gemini A Ultra using Cu-Ka radiation (l = 1.5418 A)
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7-Hydroxy-2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione
(11a)
phenyl); 7.60 (1H, dd, J = 7.6, 7.6, H-7); 7.64 (1H, dd, J = 7.6,
2.0, H-8); 11.81 (1H, s, OH). 13C NMR (CDCl3, 75 MHz): d 18.7
(C-3); 27.8 (C-4); 78.5 (C-2); 113.9 (C-4a); 118.8 (C-6); 122.4 (C-
9a); 122.5 (C-6¢-phenyl); 123.7 (C-8); 127.5 (C-2¢ and C-3¢-phenyl);
131.9 (C-5a); 131.9 (C-4¢and C-5¢-phenyl); 136.7 (C-7); 143.4 (C-
1¢-phenyl); 154.7 (C-9); 161.6 (C-10a); 183.1 (C-5); 184.1 (C-10).
Anal. Calcd for C19H13BrO4: C, 59.24; H, 3.40. Found: C, 60.47;
H, 3.58.
The reaction produced 11a in 18% as a red solid. m.p.: 153–
155 ◦C. IR (KBr) nmax/cm-1 1583 and 1638 (C O); 3399 (OH);1H
NMR (CDCl3, 300 MHz): d 1.99–2.13 (1H, m, H-3a); 2.28–2.37
(1H, m, H-3b); 2.53–2.64 (1H, m, H-4a); 2.70–2.79 (1H, m, H-4b);
5.25 (1H, dd, J = 10.3, 2.8, H-2); 7.07 (1H, dd, J = 8.4, 0.9, H-8);
7.37 (1H, dd, J = 7.4, 0.9, H-10); 7.39–7.48 (5H, m, 2-phenyl); 7.53
(1H, dd, J = 8.7, 7.4, H-9); 11.99 (1H, s, OH). 13C NMR (CDCl3,
75 MHz): d 18.4 (C-4); 28.3 (C-3); 80.1 (C-2); 113.4 (C-4a); 114.0
(C-6a); 116.9 (C-10); 121.6 (C-8); 125.8 (C-2¢ and C-3¢-phenyl);
128.5 (C-6¢-phenyl); 128.7 (C-4¢ and C-5¢-phenyl); 131.8 (C-10a);
138.0 (C-9); 139.3 (C-1¢-phenyl); 162.4 (C-7); 164.4 (C-10b); 178.1
(C-5); 182.8 (C-6). Anal. Calcd for C19H14O4: C, 74.50; H, 4.61.
Found: C, 74.84; H, 4.75.
7-Hydroxy-2-p-tolyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione
(11c)
The reaction produced 11c in 24% as a red solid. m.p.: 165–
167 ◦C. IR (KBr) nmax/cm-1 1585 and 1639 (C O); 3405 (OH).1H
NMR (CDCl3, 300 MHz): d 1.99–2.12 (1H, m, H-3a); 2.25–2.34
(1H, m, H-3b); 2.40 (3H, s, CH3); 2.52–2.63 (1H, m, H-4b); 2.70–
2.79 (1H, m, H-4a); 5.21 (1H, dd, J = 10.3, 2.6, H-2); 7.06 (1H, dd,
J = 8.8, 1.2, H-8); 7.23–7.32 (4H, m, 2-p-tolyl); 7.35 (1H, dd, J =
7.6, 1.2, H-5); 7.51 (1H, dd, J = 8.8, 7.6, H-9); 11.99 (1H, s, OH). 13C
NMR (CDCl3, 75 MHz): d 18.4 (C-3); 28.2 (C-4); 21.2 (CH3); 80.1
(C-2); 113.4 (C-4a); 114.0 (C-6a); 116.9 (C-10); 121.6 (C-8); 125.8
(C-2¢ and C-3¢-phenyl); 129.4 (C-4¢ and C-5¢-phenyl); 131.8 (C-
10a); 136.3 (C-1¢-phenyl); 138.0 (C-9); 138.5 (C-6¢-phenyl); 162.5
(C-7); 164.4 (C-10b); 178.1 (C-5); 182.8 (C-6). Anal. Calcd for
C20H16O4: C, 74.99; H, 5.03. Found: C, 74.99; H, 5.32.
9-Hydroxy-2-phenyl-3,4-dihydro-2H-benzo[g]chromene-5,10-dione
(10a)
The reaction produced 10a in 32% as an orange solid. m.p.: 122–
124 ◦C. IR (KBr) nmax/cm-1 1603 and 1636 (C O); 3416 (OH).1H
NMR (CDCl3, 300 MHz): d 2.04–2.12 (1H, m, H-3a); 2.30–2.35
(1H, m, H-3b); 2.62–2.69 (1H, m, H-4a); 2.72–2.78 (1H, m, H-
4b); 5.20 (1H, dd, J = 9.6, 2.6, H-2); 7.21 (1H, dd, J = 8.1, 1.4,
H-6); 7.34–7.43 (5H, m, 2-phenyl); 7.59 (1H, dd, J = 8.1, 7.6,
H-7); 7.63 (1H, dd, 7.6, 1.4, H-8); 11.84 (1H, s, OH). 13C NMR
(CDCl3, 75 MHz): d 12.7 (C-3); 27.8 (C-4); 79.2 (C-2); 114.1 (C-
4a); 118.8 (C-6); 122.5 (C-9a); 123.7 (C-8); 125.8 (C-2¢ and C-3¢-
phenyl); 128.4 (C-6¢-phenyl); 128.7 (C-1¢-phenyl); 128.7 (C-4¢ and
C-5¢-phenyl); 131.9 (C-5a); 136.6 (C-7); 155.0 (C-9); 161.6 (C-10a);
183.3 (C-5); 184.3 (C- 10). Anal. Calcd for C19H14O4: C, 74.50; H,
4.61. Found: C, 74.51; H, 4.47.
9-Hydroxy-2-p-tolyl-3,4-dihydro-2H-benzo[g]chromene-5,10-
dione (10c)
The ◦reaction produced 10c in 36% as an orange solid. m.p.: 177–
179 C. IR (KBr) nmax/cm-1 1619 and 1644 (C O); 3433 (OH).
1H NMR (CDCl3, 300 MHz): d 2.01–2.14 (1H, m, H-3a); 2.25–
2.34 (1H, m, H-3b); 2.37 (3H, s, CH3); 2.57–2.67 (1H, m, H-4a);
2.69–2.79 (1H, m, H-4b); 5.17 (1H, dd, 9.6, 2.4, H-2); 7.19–7.22
(2H, m, H-ortho-phenyl); 7.19–7.22 (1H, m, H-6); 7.27–7.30 (2H,
m, H-meta-phenyl); 7.56–7.58 (1H, m, H-7); 7.61–7.64 (1H, m, H-
8); 11.84 (1H, s, OH). 13C NMR (CDCl3, 75 MHz): d 18.7 (C-3);
21.1 (CH3); 27.6 (C-4); 79.2 (C-2); 114.0 (C-4a); 118.7 (C-6); 122.4
(C-9a); 123.6 (C-8); 125.8 (C-2¢ and C-3¢-phenyl); 128.8 (C-4¢ and
C-5¢-phenyl); 129.3 (C-6¢-phenyl); 131.9 (C-5a); 136.1 (C-7); 138.2
(C-1¢-phenyl); 155.0 (C-9); 161.6 (C-10a); 183.3 (C-5); 184.3 (C-
10). Anal. Calcd for C20H16O4: C, 74.99; H, 5.03. Found: C, 74.33;
H, 4.99.
2-(4-bromophenyl)-7-hydroxy-3,4-dihydro-2H-benzo[h]chromene-
5,6-dione (11b)
The reaction produced 11b in 17% as a red solid. m.p.: decompose
◦
over 160 C. IR (KBr) nmax/cm-1 1589 e 1642 (C O).1H NMR
(CDCl3, 300 MHz): d 1.95–2.10 (1H, m, H-3a); 2.26–2.35 (1H, m,
H-3b); 2.52–2.63 (1H, m, H-4a); 2.70–2.79 (1H, m, H-4b); 5.20
(1H, dd, J = 10.3; 2.6, H-2); 7.08 (1H, dd, J = 8.6, 1.0, H-8); 7.27–
7.31 (2H, m, H-meta-phenyl); 7.32 (1H, dd, J = 7.6, 1.0, H-10); 7.52
(1H, dd, J = 8.3, 7.3, H-9); 7.55–7.60 (2H, m, H-ortho-phenyl). 13
C
NMR (CDCl3, 75 MHz): d 18.3 (C-3); 28.2 (C-4); 79.4 (C-2); 114.0
(C-4a); 116.8 (C-10); 121.8 (C-8; C-2¢ and C-3¢-phenyl); 122.5 (C-
6a and C-6¢-phenyl); 132.0 (C-4¢ and C-5¢-phenyl); 138.0 (C-9);
138.3 (C-10a); 139.0 (C-1¢-phenyl); 162.1 (C-7); 164.5 (C-10b);
178.1 (C-5); 182,6 (C-6). Anal. Calcd for C19H13BrO4: C, 59.24; H,
3.40. Found: C, 60.38; H, 3.47.
2-(4-chlorophenyl)-7-hydroxy-3,4-dihydro-2H-benzo[h]
chromene-5,6-dione (11d)
The ◦reaction produced 11d in 14% as a red solid. m.p.: 140–
142 C. IR (KBr) nmax/cm-1 1584 and 1638 (C O); 3412 (OH).
1H NMR (CDCl3, 300 MHz): d 1.96–2.09 (1H, m, H-3a); 2.26–
2.35 (1H, m, H-3b); 2.53–2.64 (1H, m, H-4a); 2.70–2.79 (1H, m,
H-4b); 5.22 (1H, dd, J = 10.2, 2.6, H-2); 7.08 (1H, dd, J = 8.6,
1.0, H-8); 7.34 (1H, dd, J = 7.6, 1.0, H-10); 7.34–7.38 (2H, m,
H-meta-phenyl); 7.40–7.45(2H, m, H-ortho-phenyl); 7.53 (1H, dd,
J = 8.6, 7.6, H-9); 11.98 (1H, s, OH). 13C NMR (CDCl3, 75 MHz):
d 18.3 (C-4); 28.2 (C-3); 79.4 (C-2); 113.3 (C-4a); 114.0 (C-6a);
116.8 (C-10); 121.7 (C-8); 127.2 (C-4¢ and C-5¢-phenyl); 129.0 (C-2¢
and C-3¢-phenyl); 131.6 (C-6¢-phenyl); 134.4 (C-10a); 137.8 (C-1¢-
phenyl); 138.0 (C-9); 162.1 (C-7); 164.5 (C-10b); 178.1 (C-5); 182.6
2-(4-bromophenyl)-9-hydroxy-3,4-dihydro-2H-benzo[g] chromene-
5,10-dione (10b)
The reaction produced 10b in 29% as an orange solid. m.p.: 186–
189 ◦C. IR (KBr) nmax/cm-1 1615 and 1643 (C O); 3192 (OH). 1H
NMR (CDCl3, 300 MHz): d 1.96–2.09 (1H, m, H-3a); 2.26–2.35
(1H, m, H-3b); 2.58–2.68 (1H, m, H-4a); 2.70–2.81 (1, m, H-4b);
5.14 (1H, dd, J = 9.6, 2.6, H-2); 7.21 (1H, dd, J = 7.6, 2.0, H-6);
7.27–7.31 (2H, m, H-meta-phenyl); 7.52–7.56 (2H, m, H-ortho-
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(C-6). Anal. Calcd for C19H13ClO4: C, 66.97; H, 3.85. Found: C,
66.62; H, 3.98.
NMR (CDCl3, 300 MHz): d 2.01–2.09 (1H, m, H-3a); 2.26–2.29
(1H, m, H-3b); 2.52–2.59 (1H, m, H-4a); 2.72–2.76 (1H, m, H-4b);
3.84 (3H, s, CH3); 5.18 (1H, dd, J = 10.3, 2.3, H-2); 6.95–6.97 (2H,
m, H-meta-phenyl); 7.04 (1H, dd, J = 8.8, 1.2, H-8); 7.31–7.35
(2H, m, H-ortho-phenyl); 7.33 (1H, dd, J = 7.3, 1.2, H-10); 7.49
(1H, dd, J = 8.5, 7.6, H-9); 11.96 (1H, s, OH). 13C NMR (CDCl3,
75 MHz): d18.6 (C-4); 28.1 (C-3); 53.3 (CH3); 80.0 (C-2); 113.3 (C-
4a); 114.0 (C-6a); 114.1 (C-4¢and C-5¢-phenyl); 116.9 (C-10); 121.6
(C-8); 127.4 (C-2¢ and C-3¢-phenyl); 131.3 (C-1¢-phenyl); 131.8 (C-
10a); 138.0 (C-9); 159.8 (C-6¢-phenyl); 162.6 (C-7); 164.4 (C-10b);
178.1 (C-5); 182.8 (C-6). Anal. Calcd for C20H16O5: C, 71.42; H,
4.79. Found: C, 71.21; H, 4.95.
2-(4-Chlorophenyl)-9-hydroxy-3,4-dihydro-2H-benzo[g]
chromene-5,10-dione (10d)
The reaction produced 10d in 39% as an orange solid. m.p.: 167–
169 ◦C. IR (KBr) nmax/cm-1 1610 and 1641 (C O); 3437 (OH). 1H
NMR (CDCl3, 300 MHz): d 1.96–2.10 (1H, m, H-3a); 2.27–2.36
(1H, m, H-3b); 2.58–2.70 (1H, m, H-4a); 2.72–2.81 (1H, m, H-4b);
5.16 (1H, dd, J = 9.6, 2.6, H-2); 7.22 (1H, dd, J = 7.6, 2.0, H-6);
7.33–7.41 (4H, m, H-Ar); 7.60 (1H, dd, J = 7.9, 7.3, H-7); 7.64
(1H, dd, J = 7.3, 2.0, H-8); 11.81 (1H, s, OH). 13C NMR (CDCl3,
75 MHz): d 18.7 (C-3); 27.8 (C-4); 78.5 (C-2); 113.9 (C-4a); 118.8
(C-6); 122.5 (C-9a); 123.7 (C-8); 127.2 (C-2¢ and C-3¢-phenyl);
128.9 (C-4¢ and C-5¢-phenyl); 131.8 (C-6¢-phenyl); 134.3 (C-5a);
136.7 (C-7); 137.6 (C-1¢-phenyl); 154.7 (C-9); 161.6 (C-10a); 183.2
(C-5); 184.3 (C-10). Anal. Calcd for C19H13ClO4: C, 66.97; H, 3.85.
Found: C, 66.94; H, 4.12.
9-Hydroxy-2-(4-methoxyphenyl)-3,4-dihydro-2H-benzo[g]
chromene-5,10-dione (10f)
The reaction produced 10f in 41% as an orange solid. m.p.: 143–
145 ◦C. IR (KBr) nmax/cm-1 1618 and 1643 (C O). H NMR
1
(CDCl3, 300 MHz): d 2.01–2.14 (1H, m, H-3a); 2.24–2.33 (1H, m,
H-3b); 2.57–2.69 (1H, m, H-4a); 2.73–2.82 (1H, m, H-4b); 3.82
(3H, s, CH3); 5.15 (1H, dd, J = 9.9, 2.6,H-2); 6.90–6.95 (2H, m,
H-meta-phenyl); 7.21 (1H, dd, J = 7.8, 1.9, H-6); 7.31–7.35 (2H, m,
H-ortho-phenyl); 7.59 (1H, dd, J = 7.8, 7.5, H-7); 7.63 (1H, dd, J =
7.5, 1.9, H-8); 11.83 (1H, s, OH). 13C NMR (CDCl3, 75 MHz): d
18.9 (C-3); 27.7 (C-4); 53.3 (CH3); 79.2 (C-2); 114.1 (C-4a); 114.2
(C-4¢ and C-5¢-phenyl); 118.8 (C-6); 122.4 (C-10a); 123.6 (C-8);
127.4 (C-2¢ and C-3¢-phenyl); 131.2 (C-1¢-phenyl); 132.1 (C-6a);
136.6 (C-7); 155.2 (C-9); 159.8 (C-6¢-phenyl); 161.7 (C-10a); 183.4
(C-5); 184.4 (C-10). Anal. Calcd for C20H16O5: C, 71.42; H, 4.79.
Found: C, 71.56; H, 5.08.
2-(4-Fluorophenyl)-7-hydroxy-3,4-dihydro-2H-benzo[h]
chromene-5,6-dione (11e)
The ◦reaction produced 11e in 24% as a red solid. m.p.: 181–
183 C. IR (KBr) nmax/cm-1 1584 and 1640 (C O); 3067 (OH).
1H NMR (CDCl3, 300 MHz): d 1.97–2.11 (1H, m, H-3a); 2.35–
2.27(1H, m, H-3b); 2.52–2.64- (1H, m, H-4a); 2.72–2.80 (1H, m,
H-4b); 5.22 (1H, dd, J = 10.2, 2.3, H-2); 7.08 (1H, dd, J = 8.6, 1.0,
H-8); 7.10–7.18 (2H, m, H-meta-phenyl); 7.34 (1H, dd, J = 7.6,
1.0, H-10); 7.37–7.43 (2H, m, H-ortho-phenyl); 7.53 (1H, dd, J =
8.6, 7.6, H-9); 11.98 (1H, s, OH). 13C NMR (CDCl3, 75 MHz): d
18.4 (C-3); 28.3 (C-4); 79.5 (C-2); 113.3 (C-4a); 114.0 (C-6a); 115.7
(J = 21.0, C-4¢ and C-5¢-phenyl); 116.8 (C-10); 121.7 (C-8); 127.7
(J = 7.7, C-2¢ and C-3¢-phenyl); 135.1 (C-10a); 135.1 (C-1¢-phenyl);
138.0 (C-9); 161.0 (C-7); 162.7 (J = 247.7, C-6¢-phenyl); 164.5 (C-
10b); 178.1 (C-5); 182.7 (C-6). Anal. Calcd for C19H13FO4: C,
70.37; H, 4.04. Found: C, 70.18; H, 4.39.
7-Hydroxy-2-methyl-2-phenyl-3,4-dihydro-2H-benzo[h]
chromene-5,6-dione (11g)
The reaction produced 11g in 18% as a red solid. m.p.: 161–
162 ◦C. IR (KBr) nmax/cm-1 1593 and 1640 (C O); 3415 (OH). 1H
NMR (CDCl3, 300 MHz): d 1.76 (3H, s, CH3); 2.04–2.13 (1H, m,
H-3a); 2.16–2.20 (1H, m, H-3b); 2.45–2.53 (1H, m, H-4a); 2.62–
2.71 (1H, m, H-4b); 7.27–7.35 (5H, m, 2-phenyl); 7.18–7.23 (1H,
2-(4-Fluorophenyl)-9-hydroxy-3,4-dihydro-2H-benzo[g]
chromene-5,10-dione (10e)
m, H-9); 7.56–7.58 (2H, m, H-8 and H-10); 11.90 (1H, s, OH). 13
C
NMR (CDCl3, 75 MHz): d 17.0 (C-3); 29.7 (CH3); 31.1 (C-4); 81.7
(C-2); 114.1 (C-4a); 118.7 (C-10); 122.6 (C-6a); 123.6 (C-8); 124.1
(C-6¢-phenyl); 127.5 (C-2¢ and C-3¢-phenyl); 128.8 (C-4¢ and C-
5¢-phenyl); 131.9 (C-10a); 136.5 (C-9); 143.4 (C-1¢-phenyl); 154.0
(C-8); 161.5 (C-10b); 183.4 (C-5); 184.5 (C-6). Anal. Calcd for
C20H16O4: C, 74.99; H, 5.03. Found: C, 74.54; H, 5.36.
The reaction produced 10e in 33% as an orange solid. m.p.: 138–
140 ◦C. IR (KBr) nmax/cm-1 1617 and 1640 (C O); 3420 (OH). 1H
NMR (CDCl3, 300 MHz): d 1.98–2.11 (1H, m, H-3a); 2.27–2.35
(1H, m, H-3b); 2.58–2.70 (1H, m, H-4a); 2.74–2.83 (1H, m, H-4b);
5.15 (1H, dd, J = 9.9, 2.6, H-2); 7.06–7.14 (2H, m, H-meta-phenyl);
7.21 (1H, J = dd, 7.6, 2.0, H-6); 7.35–7.42 (2H, m, H-ortho-phenyl);
7.59 (1H, dd, J = 7.6, 7.6, H-7); 7.64 (1H, dd, J = 7.6, 2.0, H-8);
11.81 (1H, s, OH). 13C NMR (CDCl3, 75 MHz): d18.8 (C-3); 27.9
(C-4); 78.6 (C-2); 113.9 (C-4a); 115.7 (J = 21.0; C-4¢ and C-5¢-
phenyl); 118.8 (C-6); 122.4 (C-9a); 123.7 (C-8); 127.7 (J = 7.7, C-2¢
and C-3¢-phenyl); 131.8 (C-1¢-phenyl); 134.9 (C-5a); 136.6 (C-7);
154.7 (C-9); 161.6 (C-10a); 162.6 (J = 246.6, C-6¢-phenyl); 183.2
(C-5); 184.3 (C-10). Anal. Calcd for C19H13FO4: C, 70.37; H, 4.04.
Found: C, 70.23; H, 4.32.
9-Hydroxy-2-methyl-2-phenyl-3,4-dihydro-2H-benzo[g]
chromene-5,10-dione (10g)
The reaction produced 10g in 23% as an orange solid. m.p.: 126–
127 ◦C. IR (KBr) nmax/cm-1 1611 and 1641 (C O); 3428 (OH). 1H
NMR (CDCl3, 300 MHz): d 1.75 (3H, s, CH3); 2.01–2.10 (1H, m,
H-3a); 2.15–2.26 (1H, m, H-3b); 2.39–2.46 (1H, m, H-4a); 2.56–
2.64 (1H, m, H-4b); 7.31–7.35 (5H, m, 2-phenyl); 7.11 (1H, dd,
J = 8.3, 1.3, H-8); 7.49 (1H, dd, J = 8.5, 7.6, H-7); 7.62 (1H, dd,
J = 7.9, 7.6, H-6); 11.99 (1H, s, OH). 13C NMR (CDCl3, 75 MHz):
d 16.3 (C-3); 29.3 (C-CH3); 31.8 (C-4); 82.7 (C-2); 113.5 (C-4a);
114.0 (C-9a); 116.6 (C-6); 121.7 (C-8); 124.0 (C-6¢-phenyl); 127.6
(C-2¢ and C-3¢-phenyl); 128.8 (C-4¢ and C-5¢-phenyl); 132.0 (C-5a);
7-Hydroxy-2-(4-methoxyphenyl)-3,4-dihydro-2H-benzo[h]
chromene-5,6-dione (11f)
The reaction produced 11f in 13% as a red solid. m.p.: 128–
130 ◦C. IR (KBr) nmax/cm-1 1584 and 1638 (C O); 3270 (OH). 1H
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138.1 (C-7); 143.5 (C-1¢-phenyl); 161.3 (C-9); 164.5 (C-10a); 178.0
(C-5); 182.9 (C-10). Anal. Calcd for C20H16O4: C, 74.99; H, 5.03.
Found: C, 74.47; H, 5.36.
determined by reduction of the yellow dye 3-(4,5-dimethyl-2-
thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue
formazan product, as described by Mosmann.32
Cell Membrane Disruption. This test was performed in 96-
well plates using a 2% mouse erythrocyte suspension in 0.85%
NaCl containing 10 mM CaCl2, following the method previously
described.36 The compounds were tested at concentrations ranging
from 1.95 to 250 mg mL-1. After incubation at room temperature
for 30 min and centrifugation, the supernatant was removed, and
the liberated hemoglobin was measured spectrophotometrically
at 540 nm. DMSO was used as a negative control, and Triton
X-100 (1%) was used as a positive control. After incubation at
room temperature for 60 min and centrifugation, the supernatant
was removed, and the liberated hemoglobin was measured spec-
trophotometrically at 540 nm. The EC50 is the calculated effective
concentration that induced 50% of the lysis induced by Triton
X-100.
General procedures for preparing 10h and 11h
A 600 mL reactor was loaded with 9 (2.6 mmol), paraformalde-
hyde (8 mmol), isobutylene and ethyl alcohol (150 mL). The
reaction was heated at 150 ◦C for 3 h, after which the ethyl alcohol
was evaporated under reduced pressure. The crude mixture was
extracted with ethyl acetate, and the organic layer was washed
with water, dried over anhydrous sodium sulfate, filtered and
concentrated under reduced pressure. The residual crude product
was purified via silica-gel chromatography, using a gradient
mixture of hexane and ethyl acetate.
7-hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-
dione (11h)
The reaction produced 11h in 23% as a red solid. m.p.: 150–
152 ◦C. IR (KBr) nmax/cm-1 1579 and 1639 (C O); 3424 (OH). 1H
NMR (CDCl3, 300 MHz): d 1.45 (6H, s, CH3); 1.84 (1H, dd, 6.6,
6.6, H-3); 2.45 (1H, dd, J = 6.6, 6.6, H-4); 7.05 (1H, dd, J = 8.6, 1.0,
H-8); 7.34 (1H, dd, J = 7.6, 1.0, H-10); 7.53 (1H, dd, J = 8.6, 7.6,
H-9); 11.98 (1H, s, OH). 13C NMR (CDCl3, 75 MHz): d 16.1 (C-
3); 26.7 (CH3); 31.5 (C-4); 79.2 (C-2); 112.7 (C-4a); 116.7 (C-10);
113.5 (C-6a); 121.4 (C-8); 132.3 (C-10a); 137.8 (C-9); 161.5 (C-7);
164.3 (C-10b); 178.1 (C-5); 183.1 (C-6). Anal. Calcd for C15H14O4:
C, 69.76; H, 5.46. Found: C, 69.34; H, 6.05.
Acknowledgements
This work was supported by CNPq (National Council of Re-
search of Brazil), FINEP, CAPES, PRONEX-FAPERJ grant E-
26/110.574/2010 and Fundacio´n Teo´filo Hernando (Spain). The
authors are grateful for the use of the facilities of the LDRX/UFF
and LabCri/UFMG. WCS and VFF are members of the INCT
de Processos Redox em Biomedicina funded by CNPq/FAPESP
(Brazil).
Notes and references
9-hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo[g] chromene-5,10-
dione (10h)
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The ◦reaction produced 10h in 40% as an orange solid. m.p.: 178–
180 C. IR (KBr) nmax/cm-1 1608 and 1640 (C O); 3411 (OH).
1H NMR (CDCl3, 300 MHz): d 1.44 (6H, s, CH3); 1.82 (1H, dd,
6.6, 6.6, H-3); 2.61(1H, dd, J = 6.6, 6.6, H-4); 7.19 (1H, dd, J =
7.8, 1.9, H-7); 7.56 (1H, dd, J = 7.8, 0.3, H-8); 7.60 (1H, dd, J =
1.9, 0.3 H-6); 11.84 (1H, s, OH). 13C NMR (CDCl3, 75 MHz): d
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(C-6); 122.4 (C-9a); 123.4 (C-8); 132.1 (C-5a); 136.4 (C-7); 154.1
(C-9); 161.6 (C-10a); 183.3 (C-5); 184.8 (C-10). Anal. Calcd for
C15H14O4: C, 69.76; H, 5.46. Found: C, 69.38; H, 5.73.
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